WO2012147674A1 - Procédé de production de monatine - Google Patents

Procédé de production de monatine Download PDF

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Publication number
WO2012147674A1
WO2012147674A1 PCT/JP2012/060824 JP2012060824W WO2012147674A1 WO 2012147674 A1 WO2012147674 A1 WO 2012147674A1 JP 2012060824 W JP2012060824 W JP 2012060824W WO 2012147674 A1 WO2012147674 A1 WO 2012147674A1
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aminotransferase
seq
amino acid
substitution
monatin
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PCT/JP2012/060824
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Japanese (ja)
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杉山　雅一
康彰 高倉
美加 守屋
佑介 萩原
英莉 田渕
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom

Definitions

  • the present invention relates to a method for producing monatin and the like.
  • monatin is produced by synthesizing indole-3-pyruvic acid (hereinafter referred to as “IPA” if necessary) from L-tryptophan (L-Trp), and obtaining the resulting IPA and pyrubin.
  • 4R isomer of 4- (indol-3-yl-methyl) -4-hydroxy-2-oxoglutaric acid (hereinafter referred to as “4R-IHOG” if necessary) was synthesized from the acid, and then the 4R obtained
  • the following method (conventional method (1)) for producing 2R, 4R-monatin by subjecting -IHOG to oximation reaction, reduction reaction and epicrystallization crystallization method is known (see Patent Document 2) ).
  • the aldolase step (second step) is an equilibrium reaction, it cannot always be said that a satisfactory yield is obtained.
  • An object of the present invention is to provide a method for producing 2R, 4R-monatin with good yield and low cost.
  • a process for producing 2R, 4R-monatin or a salt thereof including the following: (1) contacting L-tryptophan with a deaminase to produce indole-3-pyruvate; (2) contacting indole-3-pyruvate and pyruvate with aldolase to produce 4R-IHOG; and (3) contacting 4R-IHOG with D-aminotransferase in the presence of D-amino acid.
  • To produce 2R, 4R-monatin. [2] The method of [1], wherein the steps (1) to (3) are carried out in one reaction tank.
  • D-aminotransferase is selected from the group consisting of Achromobacter, Agrobacterium, Bacillus, Coprococcus, Geobacillus, Halothiobacillus, Lactobacillus, Oceanibalbus, Paenibacillus, Rhodobacter, Robiginata
  • Achromobacter Agrobacterium
  • Bacillus Bacillus
  • Coprococcus Geobacillus
  • Halothiobacillus Lactobacillus
  • Oceanibalbus Paenibacillus
  • Rhodobacter Rhodobacter
  • Robiginata The method according to any one of [1] to [4], which is derived from a microorganism belonging to the genus or genus Thiobacillus.
  • D-aminotransferase is selected from the group consisting of Achromobacter xylosoxidans, Agrobacterium radiobacter, Bacillus halodurans, Bacillus megaterium, Bacillus macerans, Bacillus proteinformans, Coprococcus comes, Geobacillus Spi, Geobacillus tobi, Halothiobacillus neapolitanus, Lactobacillus salivarius, Oceaniva barus Indolifex, Paenibacillus larvae, Rhodobacter sphaeroides, Robiginata biformata, or Thiobacillus denitrificans Any one of [1] to [5].
  • [7] A group in which D-aminotransferase comprises amino acid residues at positions 87, 100, 117, 145, 157, 240, 243 and 244 in the amino acid sequence represented by SEQ ID NO: 2.
  • amino acid sequence represented by SEQ ID NO: 2 one or more selected from the group consisting of amino acid residues at positions 87, 100, 117, 145, 157, 240, 243 and 244 [14] D-aminotransferase comprising a mutation of an amino acid residue.
  • [17] A polynucleotide encoding the D-aminotransferase of [14].
  • the method of the present invention can produce 2R, 4R-monatin with good yield from L-Trp, which is an inexpensive raw material.
  • the method of the present invention can also be carried out from L-Trp by carrying out a deamination reaction with a deaminase, a condensation reaction with an aldolase, and an amination reaction with a D-aminotransferase (one-pot enzyme reaction).
  • 2R, 4R-monatin can be produced in good yield.
  • the method of the present invention can further produce 2R, 4R-monatin from L-Trp in a very good yield by using an IPA-inert D-aminotransferase.
  • FIG. 1 is a diagram showing an outline of the production method of the present invention.
  • L-Trp L-tryptophan
  • IPA indole-3-pyruvic acid
  • PA pyruvic acid
  • 4R-IHOG 4R-4- (indol-3-yl-methyl) -4-hydroxy-2-oxoglutaric acid
  • 2R , 4R-monatin 2R, 4R-4- (indol-3-yl-methyl) -4-hydroxy-glutamic acid.
  • FIG. 2 is a diagram showing an example of the production method of the present invention. The abbreviations are the same as in FIG.
  • D-aminotransferase has the ability to produce 2R, 4R-monatin from 4R-IHOG in the presence of D-amino acid and does not or does not produce D-Trp from IPA A thing with low capability is preferable.
  • FIG. 3 shows a preferred example of the production method of the present invention. The abbreviations are the same as in the previous figure.
  • FIG. 4 is a diagram showing an example of the production method of the present invention.
  • D-Ala D-alanine. Other abbreviations are the same as in the previous figure.
  • FIG. 5 shows a preferred example of the production method of the present invention.
  • the abbreviations are the same as in the previous figure.
  • FIG. 6 is a diagram showing an example of the manufacturing method of the present invention. The abbreviations are the same as in the previous figure.
  • FIG. 7 is a view showing a preferred example of the production method of the present invention. The abbreviations are the same as in the previous figure.
  • FIG. 8 is a diagram showing an example of the production method of the present invention.
  • FIG. 9 is a diagram showing an example of the production method of the present invention.
  • D-Asp D-aspartic acid
  • OAA oxaloacetic acid.
  • FIG. 10 is a diagram showing an example of the manufacturing method of the present invention.
  • FIG. 11 is a diagram showing an example of the production method of the present invention.
  • FIG. 12 shows a preferred example of the production method of the present invention.
  • the abbreviations are the same as in the previous figure.
  • FIG. 13 is a diagram showing the transition of D-Trp and 2R, 4R-monatin.
  • D-Trp D-tryptophan
  • RR-monatin 2R, 4R-monatin [2R, 4R-4- (indol-3-yl-methyl) -4-hydroxy-glutamic acid].
  • FIG. 14 is a diagram showing the transition of the indole compound over time. The abbreviations are the same as in the previous figure. The abbreviations are the same as in the previous figure.
  • FIG. 15 is a diagram showing the transition of the indole compound over time. The abbreviations are the same as in the previous figure.
  • the present invention provides a process for producing 2R, 4R-monatin or a salt thereof.
  • the method of the present invention includes the following (1) to (3) (see FIG. 1). (1) Contacting L-tryptophan (L-Trp) with a deaminase to produce indole-3-pyruvate (IPA) (deamination reaction); (2) contacting indole-3-pyruvate (IPA) and pyruvate (PA) with aldolase to produce 4R-IHOG (condensation reaction); and (3) in the presence of D-amino acid, 4R -Contacting IHOG with D-aminotransferase to produce 2R, 4R-monatin (amination reaction).
  • the reactions (1) to (3) are performed using, for example, an enzyme, an enzyme-producing bacterium, or a combination thereof.
  • the above-mentioned deamination reaction, condensation reaction and amination reaction may proceed separately or in parallel. These reactions may be carried out in one reaction tank (eg, one-pot enzyme reaction). When performing these reactions in one reaction vessel, these reactions can be performed by adding the substrate and enzyme sequentially or simultaneously.
  • these reactions can be performed by adding the substrate and enzyme sequentially or simultaneously.
  • L-Trp and deaminase or its producing bacterium (1) L-Trp and deaminase or its producing bacterium, (2) pyruvate and aldolase or its producing bacterium And (3) D-amino acid and D-aminotransferase or a bacterium producing the same may be added sequentially or simultaneously in one reaction vessel.
  • the producing bacterium may produce two or more enzymes selected from the group consisting of deaminase, aldolase and D-aminotransferase.
  • Deamination reaction refers to an enzyme capable of producing IPA from L-Trp.
  • the production of IPA from L-Trp is essentially the conversion of the amino group (—NH 2 ) of L-Trp to an oxo group ( ⁇ O). Therefore, an enzyme that catalyzes this reaction may be referred to as another name such as deaminase, oxidase, dehydrogenase, or L-aminotransferase.
  • deaminase is intended to mean any enzyme capable of producing IPA from L-Trp, and is an alias enzyme that catalyzes the reaction of producing IPA from L-Trp (eg, deaminase, oxidase) , Dehydrogenase or L-aminotransferase) are also included in the “deaminase”.
  • Examples of a method for producing IPA from L-Trp using a deaminase capable of acting on L-Trp to produce IPA or its producing bacterium include a method disclosed in International Publication No. 2009/0283338.
  • the general formula for the reaction catalyzed by deaminase includes the following ⁇ formula: amino acid + H 2 O ⁇ 2-oxo acid + NH 3 >.
  • Examples of a method for producing IPA from L-Trp using an oxidase capable of producing IPA by acting on L-Trp or its producing bacteria include, for example, US Pat. No. 5,002,963, John A. et al. Examples include those disclosed in Duerre et al. (Journal of Bacteriology 1975, vol 121, No. 2, p656-663), Japanese Patent Application Laid-Open No. 57-146573, International Publication No. 2003/056026, International Publication No. 2009/0283338. It is done.
  • the general formula of the reaction catalyzed by oxidase includes the following ⁇ formula: amino acid + O 2 + H 2 O ⁇ 2-oxo acid + H 2 O 2 + NH 3 >.
  • a hydrogen peroxide-degrading enzyme such as catalase may be added to the reaction solution for the purpose of suppressing decomposition of the compound by hydrogen peroxide produced as a by-product.
  • L-amino acid dehydrogenase can also be used as a method for producing IPA from L-Trp using a dehydrogenase capable of acting on L-Trp to produce IPA or its producing bacteria.
  • L-amino acid dehydrogenase used in the reaction see, for example, Toshihisa Ohshima and Kenji Soda, Stereoselective biocatalysis: amino acid dehydrogenases and thereration applications. Examples include a method using an enzyme disclosed in Stereoselective Biocatalysis (2000), 877-902.
  • the general formula of the reaction catalyzed by dehydrogenase includes the following ⁇ formula: L-amino acid + NAD (P) + H 2 O ⁇ 2-oxo acid + NAD (P) H + NH 3 >.
  • Examples of a method for producing IPA from L-Trp using L-aminotransferase capable of producing IPA by acting on L-Trp or its producing bacteria include, for example, East German Patent DD 297190, JP-A-59-95894. And the method disclosed in International Publication No. WO2003 / 091396 and US Patent Application Publication No. 2005/0282260.
  • the general formula of the reaction catalyzed by L-aminotransferase includes the following ⁇ formula:
  • deaminase used in the deamination reaction
  • enzymes disclosed in International Publication No. 2003/091396 and US Patent Application Publication No. 2005/0244937 may be used.
  • the following enzymes are used.
  • the following enzymes are abbreviated as deaminases such as deaminase, oxidase, dehydrogenase, L-aminotransferase and the like as long as they can produce IPA from L-Trp.
  • EC 2.6.1.17 Tryptophan aminotransferase that converts L-tryptophan and 2-oxoglutarate to indole-3-pyruvate and L-glutamate (L-phenylalanine-2-oxoglutarate aminotransferase, tryptophan transaminase, 5-hydroxytryptophan-ketoglutarate transaminase, hydroxytryptophan aminotransferase, L-tryptophan aminotransferase, L-tryptophan transaminase, and L-tryptophan: also called 2-oxoglutarate aminotransferase); EC 1.4.1.19: Tryptophan dehydrogenase (NAD (P) -L-tryptophan dehydrogenase, L, which converts L-tryptophan and NAD (P) to indole-3-pyruvate and NH3 and NAD (P) H Tryptophan dehydrogenase,
  • L- amino acid oxidase Vipera lebetine (sp P81375), Ophiophagus hannah (sp P81383), Agkistrodon rhodostoma (sp P81382), Crotalus atrox (sp P56742), Burkholderia cepacia, Arabidopsis thaliana, Caulobacter cresentus, Chlamydomonas reinltardtii, Mus musculus , Pseudomonas syringae, and Rhodococcus str.
  • Tryptophan oxidase is, for example, Coprinus sp.
  • SF-1 root vegetables with root-knot disease (Chinese cabbage), Arabidopsis thaliana, and mammals are known.
  • tryptophan dehydrogenase is known, for example, from spinach, Pisum sativum, Prosopis juliflora, bean, mesquite, wheat, corn, tomato, tobacco, Chromobacterium violaceum, and Lactobacilli.
  • the contact of L-Trp with a deaminase causes L-Trp and a deaminase (extracted enzyme) extracted from a deaminase-producing bacterium to coexist in the reaction solution.
  • a deaminase extracted enzyme
  • the deaminase-producing bacterium include a bacterium that naturally produces a deaminase and a transformant that expresses the deaminase.
  • the extracted enzyme include a purified enzyme, a crude enzyme, an enzyme-containing fraction prepared from the producer, and a crushed product and a lysate of the producer.
  • the contact of L-Trp with a deaminase can be achieved by allowing L-Trp and a deaminase-producing bacterium to coexist in a reaction solution (eg, a culture solution).
  • a reaction solution eg, a culture solution
  • the reaction solution for the deamination reaction is not particularly limited as long as the target reaction proceeds.
  • water or a buffer solution is used.
  • the buffer solution include Tris buffer solution, phosphate buffer solution, carbonate buffer solution, borate buffer solution, and acetate buffer solution.
  • a culture solution may be used as a reaction solution.
  • Such a culture solution can be prepared using, for example, a medium described later.
  • the pH of the reaction solution for the deamination reaction is not particularly limited as long as the target reaction proceeds.
  • the pH is 5 to 10, preferably 6 to 9, and more preferably 7 to 8.
  • the reaction temperature for the deamination reaction is not particularly limited as long as the target reaction proceeds, but is, for example, 10 to 50 ° C., preferably 20 to 40 ° C., more preferably 25 to 35 ° C.
  • the reaction time for the deamination reaction is not particularly limited as long as it is sufficient to produce IPA from L-Trp. For example, it is 2 to 100 hours, preferably 4 to 50 hours, more preferably 8 to 25 hours.
  • aldolase refers to an enzyme capable of producing 4R-IHOG from IPA and PA by aldol condensation.
  • a method for producing 4R-IHOG by condensing IPA and PA with aldolase is described in, for example, International Publication No. 2003/056026, Japanese Patent Application Laid-Open No. 2006-204285, US Patent Application Publication No. / 103989.
  • these methods can be used to prepare 4R-IHOG from IPA and PA.
  • aldolase used in the condensation reaction
  • enzymes disclosed in International Publication No. 2003/091396 and US Patent Application Publication No. 2005/0244937 may be used.
  • the following enzymes are used.
  • the following enzyme is abbreviated as aldolase as mentioned above.
  • such an enzyme includes the polypeptide (EP1045-029) EC 4.1.1.3.16, 4-hydroxy-2-oxoglutarate aldolase, 4-hydroxy-2-oxoglutarate glyoxylate-lyase, also called 2-oxo-4-hydroxyglutarate aldolase or KHG aldolase), And the polypeptide 4-hydroxy-4-methyl-2-oxoglutarate aldolase (also referred to as EC 4.1.3.17, 4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase or ProA aldolase) Can be mentioned.
  • polypeptide EP1045-029
  • EC 4.1.1.3.16 4-hydroxy-2-oxoglutarate aldolase
  • 4-hydroxy-2-oxoglutarate glyoxylate-lyase also called 2-oxo-4-hydroxyglutarate aldolase or KHG aldolase
  • polypeptide 4-hydroxy-4-methyl-2-oxoglutarate aldolase also
  • the contact of IPA and PA with an aldolase can be achieved by allowing IPA and PA and an aldolase (extracted enzyme) extracted from an aldolase-producing bacterium to coexist in the reaction solution.
  • aldolase-producing bacteria include bacteria that naturally produce aldolase, and transformants that express aldolase.
  • the extracted enzyme include a purified enzyme, a crude enzyme, an enzyme-containing fraction prepared from the producer, and a crushed product and a lysate of the producer.
  • contact of IPA and PA with aldolase can be achieved by allowing IPA and PA and aldolase-producing bacteria to coexist in a reaction solution (eg, culture solution).
  • a reaction solution eg, culture solution
  • IPA used for the preparation of 4R-IHOG is an unstable compound. Therefore, the condensation of IPA and PA may be performed in the presence of an IPA stabilizing factor.
  • the IPA stabilizing factor include superoxide dismutase (eg, see International Publication No. 2009/0283338) and mercaptoethanol (see, eg, International Publication No. 2009/0283338).
  • superoxide dismutase eg, see International Publication No. 2009/0283338
  • mercaptoethanol see, eg, International Publication No. 2009/0283338
  • transformant that expresses superoxide dismutase is disclosed in International Publication No. 2009/0283338, such a transformant may be used in the method of the present invention.
  • reaction solution such as the reaction solution, temperature, pH, and time for the condensation reaction can be appropriately set as long as the target reaction can proceed, but may be the same as the conditions described for the deamination reaction, for example.
  • D-aminotransferase refers to an enzyme that can transfer the amino group of a D-amino acid to 4R-IHOG to produce 2R, 4R-monatin.
  • a method for producing 2R, 4R-monatin by transferring the amino group of a D-amino acid to 4R-IHOG by D-aminotransferase is disclosed in, for example, WO 2004/053125. Therefore, in the present invention, these methods can be used to prepare 2R, 4R-monatin from 4R-IHOG in the presence of D-amino acid.
  • D-aminotransferase used in the amination reaction
  • enzymes disclosed in International Publication No. 2003/091396 and US Patent Application Publication No. 2005/0244937 may be used.
  • the following enzymes are used.
  • the following enzyme is abbreviated as D-aminotransferase as long as it can transfer the amino group of D-amino acid to 4R-IHOG to produce 2R, 4R-monatin.
  • EC 2.6.1.17 Tryptophan aminotransferase EC 1.4.1.19: Tryptophan dehydrogenase EC 1.4.99.1: D-amino acid dehydrogenase EC 1.4.1.2-4 : Glutamate dehydrogenase ⁇ EC 1.4.1.20: Phenylalanine dehydrogenase ⁇ EC 2.6.1.18: Tryptophan-phenylpyruvate transaminase ⁇ EC 2.6.1.1: Aspartate aminotransferase ⁇ EC 2.6 1.5: Tyrosine (aromatic) aminotransferase EC 2.6.1. -: Aminotransferase family. Examples thereof include D-tryptophan aminotransferase and D-alanine aminotransferase.
  • the contact of 4R-IHOG with D-aminotransferase in the presence of D-amino acid is obtained by extracting 4R-IHOG, and D-aminotransferase extracted from D-aminotransferase-producing bacteria (extract enzyme).
  • extract enzyme D-aminotransferase extracted from D-aminotransferase-producing bacteria
  • Examples of the D-aminotransferase-producing bacterium include bacteria that naturally produce D-aminotransferase and transformants that express D-aminotransferase.
  • examples of the extracted enzyme include a purified enzyme, a crude enzyme, an enzyme-containing fraction prepared from the producer, and a crushed product and a lysate of the producer.
  • the contact of 4R-IHOG with D-aminotransferase in the presence of D-amino acid causes the 4R-IHOG and D-aminotransferase producing bacteria to react with the reaction solution containing D-amino acid (for example, it can be achieved by coexisting in the culture medium.
  • the type of D-amino acid is not particularly limited as long as it is a D-amino acid whose amino group can be transferred to 4R-IHOG, which is the target substrate, by D-aminotransferase.
  • various D-amino acids such as D- ⁇ -amino acids are known.
  • D-amino acids include D-aspartic acid, D-alanine, D-lysine, D-arginine, D-histidine, D-glutamic acid, D-asparagine, D-glutamine, D-serine.
  • D-threonine D-tyrosine, D-cysteine
  • D-valine D-leucine
  • D-isoleucine D-proline
  • D-phenylalanine D-methionine
  • D-tryptophan D-threonine, D-tyrosine, D-cysteine, D-valine, D-leucine, D-isoleucine, D-proline, D-phenylalanine, D-methionine, D-tryptophan.
  • reaction solution for the amino reaction
  • temperature for the amino reaction
  • pH a pH
  • time for the amino reaction can be appropriately set as long as the target reaction can proceed.
  • the conditions may be the same as those described for the deamination reaction.
  • the reaction solution for the amino reaction may further contain pyridoxal phosphate (PLP) as a coenzyme.
  • PRP pyridoxal phosphate
  • the D-aminotransferase used for the amination reaction has the ability to produce 2R, 4R-monatin from 4R-IHOG in the presence of D-amino acid, and produces D-Trp from IPA. It may not have the ability or may have a low ability to generate (FIG. 2). Such properties of D-aminotransferase can also be expressed as a ratio of 4R-IHOG amination activity to IPA amination activity.
  • a D-aminotransferase having a lower IPA amination activity than the 4R-IHOG amination activity more preferably a D-aminotransferase having an IPA amination activity that is 1/10 of the 4R-IHOG amination activity, and even more
  • a D-aminotransferase having an IPA amination activity of 1/100 or less of the 4R-IHOG amination activity particularly preferably a D-aminotransferase having no IPA amination activity can be used.
  • the above-mentioned D-aminotransferase may be a protein derived from a microorganism such as bacteria, actinomycetes, or yeast. Microorganisms are classified according to methods well known in the art, for example, NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwwtc.g? 91347). Examples of microorganisms from which such D-aminotransferases are derived include the genera Achromobacter, Agrobacterium, Bacillus, Coprococcus and Geobacillus.
  • Halothiobacillus genus Lactobacillus genus, Oceanibulbus genus, Paenibacillus genus, Rhodobacter genus Is mentioned.
  • examples of such microorganisms include Achromobacter xylosoxidans, Agrobacterium radiobacter, Bacillus halodurans, Bacillus megaterans Bacillus megaterium, Bacillus macerans, Bacillus proteinformans, Barodurans, Coprococus gespires, Coprococcus commosges.
  • Chirusu-Toebi (Geobacillus toebii), halo thio Bacillus Neaporitanusu (Halothiobacillus neapolitanus), Lactobacillus salivarius (Lactobacillus salivarius), L'Oceanografic Nibaru bus and India re-fetch box (Oceanibulbus indolifex), Paenibacillus larvae (Paenibacillus larvae), Rhodobacter sphaeroides ( Examples include Rhodobacter sphaeroides), Robiginitalea biformata, and Thiobacillus denitrificans.
  • the D-aminotransferase described above can also be a natural protein or an artificial mutant protein.
  • Such D-aminotransferase can be screened from any D-aminotransferase expressed by microorganisms such as bacteria, actinomycetes and yeast.
  • D-aminotransferase examples include SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 Or 80% or more, preferably 90% or more, more preferably 95%, particularly preferably 98% or 99% or more homology (eg, similarity,) to the amino acid sequence represented by SEQ ID NO: 86 And a protein having an amino acid sequence having identity) and having D-amin
  • Such a D-aminotransferase also comprises a) introducing one or more amino acid mutations into any D-aminotransferase to produce a D-aminotransferase mutant, and b) of the produced D-aminotransferase mutant.
  • D-amino acid in the presence of D-amino acid, it retains the ability to produce 2R, 4R-monatin from 4R-IHOG, and has no or low ability to produce D-Trp from IPA It can be obtained by selecting one.
  • D-aminotransferase variants include SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, Sequence number 58, sequence number 60, sequence number 62, sequence number 64, sequence number 66, sequence number 68, sequence number 70, sequence number 72, sequence number 74, sequence number 76, sequence number 78, sequence number 80, sequence number 82, an amino acid sequence comprising a mutation (eg, deletion, substitution, addition and insertion) of one or several amino acid residues in the amino acid sequence represented by the amino acid sequence represented by SEQ ID NO: 84 or 86 And a protein having D-aminotransferase activity.
  • a mutation eg, deletion, substitution, addition and insertion
  • the mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions.
  • the term “one or several” indicates a range that does not significantly impair the three-dimensional structure and activity of the protein.
  • the number indicated by the term “one or several” in the case of protein is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5.
  • Such a mutation may be caused by a naturally occurring mutation (mutant or variant) based on individual differences, species differences, and the like of microorganisms carrying a gene encoding D-aminotransferase.
  • the D-aminotransferase variant also comprises amino acid residues at positions 87, 100, 117, 145, 157, 240, 243 and 244 in the amino acid sequence represented by SEQ ID NO: 2.
  • the amino acid residue present at the position corresponding to the above position of SEQ ID NO: 2 can be grasped by comparing the amino acid sequences by alignment.
  • the mutation of the amino acid residue in the amino acid sequence represented by SEQ ID NO: 2 or the like may be substitution of an amino acid residue selected from the group consisting of: i) Replacement of histidine at position 87 with arginine: ii) substitution of asparagine at position 100 with threonine; iii) substitution of lysine at position 117 with arginine or glutamine; iv) substitution of isoleucine at position 145 with valine; v) substitution of lysine at position 157 with arginine, glutamine or threonine; vi) substitution of serine at position 240 with threonine vii) substitution of serine at position 243 with asparagine; and viii) substitution of serine at position 244 with lysine
  • Sequence number 2 Sequence number 8
  • Sequence number 44 Sequence number 46, Sequence number 48, Sequence number 50, Sequence number 52, Sequence number 54, Sequence number 56, Sequence number 58, Sequence number 60, Sequence number 62, Sequence number 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, or SEQ ID NO: 86
  • the D-aminotransferase mutant containing the amino acid residue mutation at the above position in the amino acid sequence includes I) SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, S
  • the amino acid residue at the above position is mutated (hereinafter abbreviated as mutated amino acid sequence, if necessary) That) the high homology (e.g., similarity, consist identity) amino acid sequence having and having a D- aminotransferase activity.
  • D-aminotransferase activity refers to the activity of transferring the amino group of a D-amino acid to 4R-IHOG, which is a target substrate, to produce 2R, 4R-monatin, which is a target compound having an amino group. .
  • D-aminotransferase it is 80% or more, preferably 90% or more, more preferably 95% with respect to the mutated amino acid sequence (mutation of one or more amino acid residues at the above positions is preserved).
  • % Particularly preferably 98% or more or 99% or more of an amino acid sequence having homology (eg, similarity, identity) and having D-aminotransferase activity.
  • the homology between the amino acid sequence and the base sequence can be determined by, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) or FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Can be determined. Since programs called BLASTP and BLASTN have been developed based on this algorithm BLAST (see http://www.ncbi.nlm.nih.gov), these programs are used with default settings, and amino acid sequences and You may calculate the homology of a base sequence.
  • a D-aminotransferase variant is a mutation of one or several amino acid residues (eg, deletion, substitution, addition and addition) in the mutated amino acid sequence (conversion of one or more amino acid residues at the above positions is conserved). It may be a protein consisting of an amino acid sequence containing an insertion) and having D-aminotransferase activity. The mutation of one or several amino acid residues may be introduced into one region in the amino acid sequence, but may be introduced into a plurality of different regions. The term “one or several” indicates a range that does not significantly impair the three-dimensional structure and activity of the protein.
  • the number indicated by the term “one or several” in the case of protein is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5.
  • Such a mutation may be caused by a naturally occurring mutation (mutant or variant) based on individual differences, species differences, and the like of microorganisms carrying a gene encoding D-aminotransferase.
  • the D-aminotransferase mutant may have a purification tag such as a histidine tag.
  • the position of the amino acid residue to be mutated in the amino acid sequence is obvious to those skilled in the art. Specifically, those skilled in the art 1) compare the amino acid sequences of a plurality of proteins having the same type of activity (eg, the amino acid sequence represented by SEQ ID NO: 2 and the amino acid sequences of other L-aminotransferases), 2) reveal the relatively conserved areas and the relatively unconserved areas, then 3) function from the relatively conserved areas and the relatively unconserved areas, respectively. It is possible to predict regions that can play an important role in the region and regions that cannot play an important role in the function, so that the correlation between structure and function can be recognized. Therefore, those skilled in the art can specify the position of the amino acid residue to be mutated in the amino acid sequence of L-aminotransferase.
  • the substitution of the amino acid residue may be a conservative substitution.
  • conservative substitution refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art.
  • such families include amino acids having basic side chains (eg, lysine, arginine, histidine), amino acids having acidic side chains (eg, aspartic acid, glutamic acid), amino acids having uncharged polar side chains (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain Amino acids (eg, threonine, valine, isoleucine), amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine), amino acids having side groups containing hydroxyl groups (eg, alcoholic, phenolic) ( Example, serine, thread Nin, tyrosine), and amino acids (e.g.
  • the conservative substitution of amino acids is a substitution between aspartic acid and glutamic acid, a substitution between arginine and lysine and histidine, a substitution between tryptophan and phenylalanine, and between phenylalanine and valine. Or a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.
  • the D-aminotransferase variants are SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, Alternatively, it may be a protein encoded by DNA that hybridizes under stringent conditions with a base sequence complementary to the base sequence represented by SEQ ID NO: 85 and having D-aminotransferase activity.
  • “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify such conditions, for example, polynucleotides having high homology (eg, identity), for example, 80%, preferably 90% or more, more preferably 95 %, Particularly preferably 98% or more of the polynucleotides having a homology hybridize, and polynucleotides having a lower homology do not hybridize. Specifically, such conditions include hybridization at about 45 ° C. in 6 ⁇ SSC (sodium chloride / sodium citrate), followed by 50 ⁇ 0.2 ⁇ SSC in 0.1% SDS. One or more washings at ⁇ 65 ° C. may be mentioned.
  • SSC sodium chloride / sodium citrate
  • the D-aminotransferase used in the amination reaction also has the ability to produce 2R, 4R-monatin from 4R-IHOG in the presence of D-amino acid, and D-alanine (D-Ala) from PA May not have the ability to generate or may have a low ability to generate (FIGS. 4 and 6).
  • D-aminotransferase can also be expressed as a ratio of 4R-IHOG amination activity to PA amination activity.
  • a D-aminotransferase having a PA amination activity lower than that of 4R-IHOG amination activity more preferably a D-aminotransferase having a PA amination activity of 1/10 of 4R-IHOG amination activity, and even more
  • a D-aminotransferase having a PA amination activity of 1/100 or less of the 4R-IHOG amination activity particularly preferably a D-aminotransferase having no PA amination activity can be used.
  • D-aminotransferase By using such D-aminotransferase, the production of D-Ala from PA is suppressed and the production of 4R-IHOG from IPA and PA is promoted, so that 2R, 4R-monatin is produced in good yield. It can be manufactured (FIGS. 4 and 6).
  • Such a D-aminotransferase has the ability to produce 2R, 4R-monatin from 4R-IHOG in the presence of D-amino acid and does not have the ability to produce D-Trp from IPA, Alternatively, it can be obtained in the same manner as the above-mentioned D-aminotransferase having a low ability to produce.
  • the D-aminotransferase used in the amination reaction has the ability to produce 2R, 4R-monatin from 4R-IHOG and the ability to produce D-Trp from IPA in the presence of D-amino acid. It does not have or has a low ability to produce, and does not have the ability to produce D-alanine (D-Ala) from PA or has a low ability to produce.
  • D-Ala D-alanine
  • the production method of the present invention comprises decomposing a keto acid (R-COCOOH) produced from a D-amino acid (eg, D- ⁇ -amino acid) by the action of D-aminotransferase by contacting with decarboxylase.
  • R-COCOOH keto acid
  • D-amino acid eg, D- ⁇ -amino acid
  • decarboxylase e.g. D-amino acid
  • FIG. 8 decomposing a keto acid (R-COCOOH) produced from a D-amino acid (eg, D- ⁇ -amino acid) by the action of D-aminotransferase by contacting with decarboxylase.
  • the decarboxylase used in the present invention is an enzyme that catalyzes the decarboxylation reaction of keto acid.
  • Decarboxylation by decarboxylase can be irreversible.
  • Various enzymes are known as decarboxylase used for irreversible decarboxylation of keto acid. For example, Pseudomonas stutzeri oxaloacetate decarboxylase (Arch Biochem Biophys., 365) 17-24, 1999), and Zymomonas mobilis-derived pyruvate decarboxylase (Applied Microbiology and Biotechnology, 17, 152-157, 1983).
  • the production method of the present invention comprises contacting oxaloacetate (OAA) produced from D-aspartate (D-Asp) by the action of D-aminotransferase with oxaloacetate decarboxylase, and then adding pyruvate. Generating (PA) (FIG. 9).
  • OAA oxaloacetate
  • PA pyruvate
  • D-aminotransferase When D-Asp is used as a D-amino acid that is one of the substrates in the amination reaction, D-aminotransferase has a substrate specificity for D-Asp, a substrate specificity for D-Trp, and a substrate for D-Ala. It may be higher than specificity or substrate specificity for D-Trp and D-Ala (FIGS. 2, 4, 6).
  • a reaction that generates 2R, 4R-monatin from 4R-IHOG is a reaction that generates D-Trp from IPA, and It is considered that the reaction proceeds more easily than the reaction of producing D-Ala from PA.
  • the oxaloacetate decarboxylase used in the present invention is an enzyme that produces PA by catalyzing the decarboxylation reaction of OAA.
  • the decarboxylation reaction with oxaloacetate decarboxylase can be irreversible.
  • Various enzymes are known as oxaloacetate decarboxylase used for irreversible decarboxylation of OAA.
  • oxaloacetate decarboxylase examples include, for example, Pseudomonas stutzeri oxaloacetate decarboxylase (Arch Biochem Biophys., 365, 17-24, 1999), Klebsiella aerogenes (Klebsiella aerogenes) Examples include acetate decarboxylase (FEBS Lett., 141, 59-62, 1982), and oxaloacetate decarboxylase (Biochim Biophys Acta., 957, 301-311, 1988) derived from Sulfolobus solfatricus.
  • decarboxylase When decarboxylase is used in the production of 2R, 4R-monatin from 4R-IHOG, the contact of the keto acid produced from the D-amino acid with the decarboxylase was extracted from the keto acid and the decarboxylase producer This can be achieved by allowing a decarboxylase (extracting enzyme) or a decarboxylase-producing bacterium to coexist in a reaction solution (eg, culture solution).
  • a decarboxylase extracting enzyme
  • a decarboxylase-producing bacteria include bacteria that naturally produce decarboxylase and transformants that express decarboxylase.
  • the extracted enzyme include a purified enzyme, a crude enzyme, an enzyme-containing fraction prepared from the producer, and a crushed product and a lysate of the producer.
  • D-aminotransferase and decarboxylase may be provided in the reaction in the following manner: .
  • the D-aminotransferase and decarboxylase producing bacterium may be a transformant.
  • a transformant is obtained by introducing i) a D-aminotransferase expression vector into a decarboxylase producing bacterium, and ii) introducing the decarboxylase expression vector into a D-aminotransferase producing bacterium.
  • D-aminotransferase and decarboxylase expression vectors include i ′) a first polynucleotide encoding D-aminotransferase, and a first promoter operably linked to the first polynucleotide.
  • Including expression Tar vector capable of expressing the polycistronic mRNA
  • the first polynucleotide encoding D-aminotransferase may be located upstream or downstream of the second polynucleotide encoding decarboxylase.
  • the production method of the present invention may further comprise contacting L-amino acid with racemase to produce D-amino acid (FIG. 10).
  • the racemase used in the present invention is an enzyme that converts L-amino acids into D-amino acids.
  • a method for producing a D-amino acid from an L-amino acid by racemase is described in, for example, Kuniki Kino et al. , Synthesis of DL-tryptophan by modified broadcast specificity amino acid racemass from Pseudomonas putida IFO 12996. Applied Microbiology and Biotechnology (2007), 73 (6), 1299-1305, Tohru Yoshimura et al.
  • L-amino acids such as L- ⁇ -amino acids are known as L-amino acids.
  • L-amino acids include L-aspartic acid, L-alanine, L-lysine, L-arginine, L-histidine, L-glutamic acid, L-asparagine, L-glutamine, L-serine, L- Examples include threonine, L-tyrosine, L-cysteine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-methionine, and L-tryptophan. Since the D-amino acid used in the amination reaction is preferably D-Asp, the L-amino acid is preferably L-Asp.
  • contact of L-amino acid with racemase can be achieved by allowing L-amino acid and racemase (extracted enzyme) extracted from racemase-producing bacteria to coexist in the reaction solution.
  • racemase-producing bacteria include bacteria that naturally produce racemase and transformants that express racemase.
  • the extracted enzyme include a purified enzyme, a crude enzyme, an enzyme-containing fraction prepared from the producer, and a crushed product and a lysate of the producer.
  • the contact of L-amino acid with racemase can be achieved by allowing L-amino acid and racemase-producing bacteria to coexist in a reaction solution (eg, culture solution).
  • a reaction solution eg, culture solution
  • D-aminotransferase and racemase are used in the production of 2R, 4R-monatin from 4R-IHOG
  • the D-aminotransferase and racemase can be synthesized in a manner similar to that described above for D-aminotransferase and decarboxylase. , May be provided in the reaction solution.
  • the production method of the present invention may include the presence of a D-amino acid dehydrogenase in the reaction vessel in order to convert D-Trp by-produced during the reaction back into IPA (FIGS. 3, 5, and 7).
  • the D-amino acid dehydrogenase used in the present invention is an enzyme that converts a D-amino acid into a corresponding keto acid.
  • Examples of D-amino acid dehydrogenases include Kavittha Vedha-Peters et al. , Creation of a Broad-Range and Highly Stereoselective D-Amino Acid Dehydrogenase for the One-Step Synthesis of D-Amino Acids.
  • a transformant when used as a producing bacterium of the target enzyme (eg, deaminase, aldolase, D-aminotransferase, decarboxylase, racemase), A transformant can be prepared by preparing an expression vector for a target enzyme and then introducing the expression vector into a host.
  • a transformant expressing the D-aminotransferase mutant of the present invention is obtained by preparing an expression vector incorporating a DNA encoding the D-aminotransferase mutant of the present invention and introducing it into an appropriate host. be able to.
  • hosts for expressing the target enzyme include various prokaryotic cells such as Escherichia coli, Escherichia coli, Corynebacterium, and Bacillus subtilis, and Saccharomyces.
  • prokaryotic cells such as Escherichia coli, Escherichia coli, Corynebacterium, and Bacillus subtilis
  • Saccharomyces Various eukaryotic cells such as Saccharomyces cerevisiae, Pichia stipitis, Aspergillus oryzae can be used.
  • the host to be transformed is as described above, but in detail about E. coli, it can be selected from Escherichia coli JM109 strain, DH5 ⁇ strain, HB101 strain, BL21 (DE3) strain, etc.
  • a method for performing transformation and a method for selecting transformants are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001/01/15) and the like.
  • a method for producing transformed E. coli and producing a predetermined enzyme using the same will be described more specifically as an example.
  • E. Promoters used for heterologous protein production in E. coli can be used.
  • T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, T5 promoter, etc. can be mentioned.
  • the vector include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pACYC177, pACYC184, pMW119, pMW118, pMW219, pMW218, pQE30, and derivatives thereof.
  • phage DNA vectors may be used.
  • an expression vector containing a promoter and capable of expressing the inserted DNA sequence may be used.
  • a terminator that is a transcription termination sequence may be linked downstream of the target enzyme gene.
  • examples of such terminators include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, and E. coli trpA gene terminator.
  • a so-called multi-copy type is preferable, and a plasmid having a replication origin derived from ColE1, such as a pUC-type plasmid, a pBR322-type plasmid, or a derivative thereof can be mentioned.
  • the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition and / or inversion.
  • the “modification” here includes modification by mutation treatment, UV irradiation, natural mutation, or the like.
  • the vector has a marker such as an ampicillin resistance gene in order to select transformants.
  • a marker such as an ampicillin resistance gene
  • an expression vector having a strong promoter is commercially available (eg, pUC system (manufactured by Takara Bio Inc.), pPROK system (manufactured by Clontech), pKK233-2 (manufactured by Clontech)).
  • the obtained expression vector is used to transform E. coli, and the E. coli is cultured, the target enzyme is expressed.
  • a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used.
  • the culture conditions and production induction conditions are appropriately selected according to the type of the marker, promoter, host fungus and the like used.
  • the target enzyme can be obtained as a crushed material and a lysate by recovering the bacteria producing the target enzyme and then crushing (eg, sonication, homogenization) or dissolving (eg, lysozyme treatment). Can do.
  • a purified enzyme, a crude enzyme, or an enzyme-containing fraction can also be obtained by subjecting such crushed material and lysate to techniques such as extraction, precipitation, filtration, and column chromatography.
  • 2R, 4R-monatin obtained by the production method of the present invention is a known separation and purification means such as concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, transfer dissolution, activated carbon treatment, ion exchange resin or synthetic adsorption resin. Isolation and purification can be carried out by combining chromatography and other treatments as necessary.
  • the compound used as a raw material in the production method of the present invention may be added to the reaction system in the form of a salt.
  • the salt of 2R, 4R-monatin produced in the present invention can be produced according to a method known per se, for example, by adding an inorganic acid or an organic acid to 2R, 4R-monatin.
  • 2R, 4R-monatin or a salt thereof may be a hydrate, and both a hydrate and a non-hydrate are included in the scope of the present invention.
  • the salt include various salts such as sodium salt, potassium salt, ammonium salt, magnesium salt and calcium salt.
  • the present invention also provides a method for producing 2R, 4R-monatin or a salt thereof in which the following two steps are carried out in one reaction vessel (FIG. 11): (1 ′) contacting indole-3-pyruvate and pyruvate with aldolase to produce 4R-IHOG; and (2 ′) 4R-IHOG to D-aminotransferase in the presence of D-amino acid. Contact to produce 2R, 4R-monatin.
  • This production method can be carried out in the same manner as the steps (2) and (3) in the production method of the present invention described above.
  • it may further include the presence of D-amino acid dehydrogenase in the reaction vessel (FIG. 12).
  • This manufacturing method may further include a step similar to the step (1) in the manufacturing method of the present invention described above.
  • Example 1 Construction of expression strain and activity measurement of DAT derived from Bacillus macerans AJ1617 1) Construction of expression strain of BMDAT PCR using a plasmid in which a dat gene (BMDAT gene) derived from Bacillus macerans AJ1617 described in International Publication No. 2004/053125 was inserted as a template Amplification was performed.
  • BMDAT gene a dat gene derived from Bacillus macerans AJ1617 described in International Publication No. 2004/053125 was inserted as a template Amplification was performed.
  • the S244K mutant enzyme is referred to as BMDAT22
  • the S243N / S244K mutant enzyme is referred to as BMDAT80.
  • the primer BmDAT-Nde-f (5′-ggatgaacgggcatATGGCATATTCATTATGGAATGATC-3 ′: SEQ ID NO: 3) and the primer BmDAT-delNde-r (5′-ttcaagttttcatCgacgtgtccccc-3 ′ sequence:
  • the primer BmDAT-delNde-f (5′-gcgggtgaacgtgcGtatgaaaactttgaa-3 ′: SEQ ID NO: 5) and the primer BmDAT-Xho-r (5′-CAAGGTTCTTctGTGATGTT PCR It was.
  • PCR amplification was performed using the two DNA fragments thus obtained as templates. Primers BmDAT-Nde-f and primer BmDAT-Xho-r were used as primers. All PCR amplifications were performed using KOD-Plus-ver. 2 (Toyobo). The obtained DNA fragment contains the BMDAT gene from which the NdeI recognition site has been deleted.
  • the conditions for PCR amplification were as follows. 1 cycle 94 ° C, 2 min 25 cycles 98 ° C, 10 sec 55 ° C, 10 sec 68 ° C, 1 min 1 cycle 68 ° C, 1 min
  • This DNA fragment was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI.
  • E. coli JM109 was transformed, the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pET22-BMDAT-His (C).
  • E. coli BL21 DE3
  • E. coli BL21 (DE3) was transformed into pET22-BMDAT-His (C) / E. coli BL21 (DE3) was obtained.
  • BMDAT with His-tag added at the C-terminus is expressed.
  • expression strains of BMDAT22 and BMDAT80 were constructed.
  • BMDAT An expression strain grown on LB-amp (100 mg / l) plates, pET22-BMDAT-His (C) / E. E. coli BL21 (DE3) cells were inoculated into 160 ml of Overnight Express TB Medium (Merck) containing 100 mg / l of ampicillin, and shake-cultured at 30 ° C. for 16 hours using a Sakaguchi flask. After completion of the culture, bacterial cells were collected from the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 100 mM NaCl, and 20 mM imidazole, and subjected to ultrasonic crushing.
  • Tris-HCl pH 7.6
  • the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
  • D-Ala- ⁇ KG ( ⁇ -ketoglutarate) activity D-Ala 100 mM, ⁇ KG-2Na 10 mM, Tris-HCl (pH 8.0) 100 mM, PLP 50 ⁇ M, NADH 0.25 mM, LDH 10 U / ml, 25 ° C.
  • the reaction was performed on a 1 ml scale for 10 minutes, and the activity was calculated from the decrease at 340 nm.
  • LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used.
  • D-Ala- ( ⁇ ) -IHOG activity D-Ala 100 mM, ( ⁇ ) -IHOG (synonymous with 4R / 4S-IHOG) 10 mM, Tris-HCl (pH 8.0) 100 mM, PLP 50 ⁇ M, 25 ° C.
  • the reaction was carried out on a 0.2 ml scale for 15 minutes, and the produced 2R, 4R-monatin (RR) and 2R, 4S-monatin (RS) were quantified by UPLC analysis and the activity was calculated.
  • As the reaction stop solution a 200 mM Na citrate solution (pH 4.5) was used.
  • the analysis conditions of UPLC were as follows.
  • D-Ala-IPA activity D-Ala 100 mM, IPA 10 mM, Tris-HCl (pH 8.0) 100 mM, PLP 50 ⁇ M, 25 ° C. After the reaction solution was prepared, the pH was adjusted to 8.0 using 1M NaOH. The reaction was performed on a 0.2 ml scale for 15 minutes, and the produced Trp was quantified by UPLC analysis and the activity was calculated. As the reaction stop solution, a 200 mM Na citrate solution (pH 4.5) was used. The analytical conditions for UPLC were as described above.
  • D-Ala- ( ⁇ ) -MHOG (4-hydroxy-4-methyl-2-oxo glutarate) activity
  • D-Ala 100 mM
  • ( ⁇ ) -MHOG (synonymous with 4R / 4S-MHOG) 10 mM
  • PLP 50 ⁇ M NADH 0.25 mM
  • LDH 10 U / ml 25 ° C.
  • the reaction was performed on a 1 ml scale for 10 minutes, and the activity was calculated from the decrease at 340 nm.
  • LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used.
  • the D-aminotransferase mutant of the present invention has the ability to produce 2R, 4R-monatin from 4R-IHOG in the presence of D-amino acid, its activity against IPA is greatly reduced. It was shown that. 2) Deamination reaction by deaminase and condensation reaction by aldolase are known as described above. Therefore, the amination reaction by the D-aminotransferase mutant of the present invention is combined with the deamination reaction and the condensation reaction, and the deamination reaction, the condensation reaction and the amination reaction are performed in one reaction vessel ( One-pot enzyme reaction), 2R, 4R-monatin can be produced from L-Trp (FIG. 1).
  • Example 2 Construction of expression strain and substrate specificity analysis of DAT derived from Bacillus proteinans AJ3844 strain Genomic DNA of Bacillus proteinformans AJ3844 strain was prepared according to a standard method, and a DNA fragment containing the DAT gene was PCR amplified using this as a template. Note that the sequence of the DAT gene derived from Bacillus proteinforms AJ3844 strain is as shown in SEQ ID NO: 7, and those skilled in the art can fully synthesize the DNA fragment by adding a necessary restriction enzyme site by PCR or the like. I can do it.
  • the primer Brevis-F-NdeI [5′-GGAATTCCCATATGCTCCTGTTAGATGGGAAATGGGTAGAAG-3 ′ (SEQ ID NO: 9)] and the primer Brevis-F-XhoI [5′-CCCTCGAGCACGAGTACACTTGTGTTTGATTGTCTGTCTGTCTGTCTGTCTGTPTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTCTGTC (TaKaRa Bio) was used.
  • the obtained DNA fragment was subjected to restriction enzyme treatment with NdeI and XhoI, and ligated with pET-22b (Novagen) similarly treated with NdeI and XhoI. With this ligation solution E.
  • E. coli JM109 was transformed, and the target plasmid was extracted from the ampicillin resistant strain. Using this plasmid, E. coli BL21 (DE3) was transformed into pET22-AJ3844DAT / E. coli BL21 (DE3) was obtained. In this expression strain, DAT having His-tag added at the C-terminus is expressed. At the time of expression, cells grown on LB-amp (100 mg / l) agar medium were inoculated into Overnight Express TB Medium (Merck) containing ampicillin 100 mg / l and shaken at 30 ° C. for 16 hours. Culture was performed.
  • AJ3844-derived DAT was expressed under three conditions of 25 ° C, 30 ° C, and 37 ° C, and the resulting C.I. F. E. was used to measure D-Asp- ⁇ -KG activity (Table 2), and the expression of DAT activity was confirmed.
  • AJ3844-derived DAT purification from the expression strain was performed.
  • the cells of E. coli BL21 (DE3) were inoculated into 100 ml of Overnight Express Instant Medium (Merck) containing 100 mg / l of ampicillin, and shake-cultured at 37 ° C. for 16 hours using a Sakaguchi flask. After completion of the culture, the cells were collected from about 200 ml of the obtained culture solution by centrifugation and purified using a His-Bind column.
  • Tris-HCl pH 7.6 20 mM, NaCl 300 mM, and Imidazole 10 mM, and sonicated.
  • the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
  • a purification scheme by His-tag affinity chromatography is shown below. The protein elution fraction was dialyzed with 20 mM Tris-HCl (pH 7.6), 10 ⁇ M PLP, and 300 mM KCl to prepare an enzyme solution.
  • D-Asp- ⁇ KG, D-Asp-PA, D-Asp- (R) -IHOG, D-Asp-MHOG and D-Asp-IPA activities were measured using the purified AJ3844DAT solution as an enzyme source.
  • Tris-HCl (pH 7.6) 20 mM, BSA 0.01% was used for dilution of the enzyme. Each activity measurement method is shown below.
  • D-Asp- ⁇ KG activity D-Asp 100 mM (adjusted to pH 8.0 with NaOH), ⁇ KG-2Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH 2 U / ml, 25 ° C. The activity was calculated from the decrease in absorbance at 340 nm.
  • D-Asp-PA activity D-Asp 100 mM (pH adjusted to 8.0 with NaOH), PA-Na 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH 2 U / ml, 25 ° C. The activity was calculated from the decrease in absorbance at 340 nm.
  • D-Asp-IPA activity D-Asp 100 mM (adjusted to pH 8.0 with NaOH), IPA 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM (pH adjusted to pH 8.0 with 1 N NaOH after preparation of reaction solution) ), And reacted at 25 ° C. for 15 minutes.
  • a sodium citrate solution (pH 4.5) was added to stop the reaction, the reaction liquid after the reaction was stopped was centrifuged, and the supernatant was subjected to UPLC analysis.
  • D-Asp- ( ⁇ ) -MHOG activity D-Asp 100 mM (adjusted to pH 8.0 with NaOH), ( ⁇ ) -MHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, NADH 0.25 mM, MDH
  • the reaction was performed at 25 ° C. with 2 U / ml, LDH 10 U / ml, and 0.2 ml, and the activity was calculated from the decrease in absorbance at 340 nm.
  • D-Asp- (R) -IHOG activity D-Asp 100 mM (adjusted to pH 8.0 with NaOH), (R) -IHOG 10 mM, PLP 50 ⁇ M, Tris-HCl (pH 8.0) 100 mM, reaction at 25 ° C. for 15 minutes Went. A sodium citrate solution (pH 4.5) was added to stop the reaction, the reaction liquid after the reaction was stopped was centrifuged, and the supernatant was subjected to UPLC analysis.
  • MDH Malic dehydrogenase from porcine heart (Sigma) was used.
  • LDH D-Lactate dehydrogenase from Leuconostoc mesenteroides (oriental yeast) was used.
  • Example 3 In Silico Screening for Highly Selective DAT The gene sequences of various DATs shown in Table 5 were subjected to GenScript Optimum Codon Optimization Analysis. A plasmid was obtained in which a synthetic DNA optimized for gene expression efficiency in E. coli was cloned into pET-22b (Novagen) treated with NdeI and XhoI. The resulting plasmid is E. coli. E. coli BL21 (DE3) was transformed to obtain various DAT expression strains expressing DAT with His-tag added to the C-terminus.
  • DAT # 19 DAT derived from Ruminococcaceae Bacterium D16
  • RR / Trp ratio D-Trp by-liveness ratio
  • 4R-monatin production activity is as high as 31.9
  • this DAT is The in silico screening candidate showed the second highest specific activity against 4R-IHOG (0.413 U / mg).
  • DAT9 and DAT19 purified enzymes were prepared.
  • Various DAT-expressing strains grown on LB-amp (100 mg / l) agar medium were inoculated into 100 ml of Overnight Express TB Medium (Merck) containing ampicillin 100 mg / l, each at a temperature of 37 ° C. for 16 hours. Shaking culture was performed using a Sakaguchi flask. After completion of the culture, bacterial cells were collected from the obtained culture broth by centrifugation, washed and suspended in 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM imidazole, and subjected to ultrasonic crushing.
  • the cell residue was removed from the disrupted solution by centrifugation, and the resulting supernatant was used as a soluble fraction.
  • the obtained soluble fraction was adsorbed to a carrier by applying to a His-tag protein purification column His TALON Superflow 5 ml Cartridge (Clontech) equilibrated with 20 mM Tris-HCl (pH 7.6), 300 mM NaCl, and 10 mM Imidazole.
  • DAT # 19 has a high RR / Trp ratio, which is the aim of this screening, and DAT # 9 does not reach DAT # 19, but has a relatively high specific activity against 4R-IHOG, and a ratio with respect to PA and MHOG.
  • the result was low activity.
  • BMDAT-22 which was previously acquired DAT, as compared with the result of measuring the specific activity against various keto acids, the DAT acquired this time had high specific activity against 4R-IHOG.
  • DAT # 9 had higher specific activity against IPA than BMDAT-22, but low specific activity against PA and MHOG.
  • DAT # 19 had high specific activity against PA and MHOG.
  • Example 4 Construction and Evaluation of Mutant Enzyme for DAT Derived from Bacillus macerans Construction of a mutant BMDAT expression plasmid by site-directed mutagenesis was performed according to the protocol of QuikChange Site-Directed Mutagenesis Kit manufactured by Stratagene. DNA primers (two pairs) designed to introduce the desired base substitution and to be complementary to each strand of the double-stranded DNA were synthesized (Table 7). Using a pET22b-BMDAT-22 produced using a pET22b vector (Novagen) having a His-tag sequence at the C-terminus, a mutant plasmid was prepared under the following reaction solution composition and PCR conditions.
  • the template plasmid pET22b-BMDAT-22 was cleaved by adding 1 ⁇ l of restriction enzyme DpnI (10 U / ⁇ l) that recognizes and cleaves methylated DNA and treats at 37 ° C. for 1-3 hours. Competent cell XL10-Gold was transformed with the resulting reaction solution. The plasmid was recovered from the transformant, the base sequence was determined, and it was confirmed that the desired base substitution was introduced.
  • the plasmid extractor PI-50 (KURABO) is used for recovering plasmids from E. coli, the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI) is used for sequencing to determine the base sequence, and the Clean SEQ Kit (ABI) is used for sample purification.
  • the obtained mutant BMDAT expression plasmid was used for E. coli.
  • E. coli JM109 (DE3) was transformed to produce a mutant BMDAT expression strain.
  • Each expression strain was ingested into 100 ml of TB-autoinducer medium (Novagen) medium containing 100 ⁇ g / ml ampicillin prepared in a 500 ml Sakaguchi flask, and reciprocally shaken at 37 ° C. and 110 rpm overnight (16 to 18 hours). .
  • the obtained culture broth was transferred to a 50 ml tube and collected by centrifugation at 6000 ⁇ g, 10 min, 4 ° C.
  • amino donor substrate for the transamination reaction 100 mM D-Asp was used, transamination reaction was performed on 10 mM of various keto acids, the amount of amino acid produced was quantified by UPLC, and the specific activity was calculated. In addition, D-Glu production activity from ⁇ KG, which is the original substrate, D-Ala by-life using PA as a substrate, and MHG by-life using MHOG as a substrate were measured. 100 mM D-Asp was used as an amino donor substrate for transamination reaction, and the specific activity against 10 mM of various keto acids was measured by a colorimetric method.
  • the target 2R, 4R-monatin / D-Trp activity ratio was improved in the mutants shown in Table 10.
  • DID-28 K157Q
  • the RR / MHG activity ratio was also improved 5 times.
  • DID-8 N100T
  • DID-8 (N100T) improved 2R, 4R-monatin activity from 0.14 to 0.44 U / mg by 3 times, while Ala by-life decreased from 35 to 16 U / mg by 1/2 times. Therefore, it is considered to be an effective mutation for suppressing Ala by-product.
  • Example 5 Examination of 2R, 4R-monatin one-pot reaction using the obtained DAT Reaction was carried out for 22 hours under the following conditions using purified DAT. The reaction was performed at 0.4 ml using a 1.5 ml tube. DAT was added 1 hour after the start of the reaction. Sampling was performed as appropriate, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa, and the filtrate was analyzed. Analysis was performed by HPLC and capillary electrophoresis. DAT evaluated BMDAT-22 in addition to DAT9 and DAT19.
  • Reaction conditions IPA 10 mM, PA-Na 100 mM, D-Asp 400 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.6, SpAld (aldolase) 30 U / ml, DAT 1 U / ml (D- Asp / 4R-IHOG activity), OAA DCase (oxaloacetate decarboxylase) 10 U / ml, SOD (superoxide dismutase) 100 U / ml, 25 ° C., 140 rpm.
  • SpAld was prepared by the following method.
  • a DNA fragment containing the SpAld gene was PCR-amplified using the plasmid DNA and ptrpSpALD described in JP-A-2006-204285 and Example 5 as a template.
  • Primers SpAld-f-NdeI (5′-GGAATTCCATATACCACCAGACGCGCTCCAA-3 ′: SEQ ID NO: 29) and primer SpAld-r-HindIII (5′-GCCCCAAGCTTTCAGTACCCCGCCAGTTCGC-3 ′: SEQ ID NO: 30) were used.
  • coli rare codons (6L-ctc, 13L-ctc, 18P-ccc, 38P-ccc, 50P-ccc, 77P-ccc, 81P-ccc, 84R-cga) are 6L-ctg, 13L-ctg, 18P-ccg, Conversion was made to 38P-ccg, 50P-ccg, 77P-ccg, 81P-ccg, 84R-cgc.
  • primer 6L-f (5'-ACCCACAGCCGCCCTGAACGGCATCATCCCG-3 ': SEQ ID NO: 31) and primer 6L-r (5'-CGGATGATGCCGTTCAGGCGCGTCTGGGGT-3': SEQ ID NO: 32) were used.
  • primer 13L-f (5'-ATCATCCGCGCTCTCTGGAAGCCGGCAAGCC-3 ': SEQ ID NO: 33) and primer 13L-r (5'-GGCTTGCCGGCTTCCCAGAGCGCGGATGATAT-3': SEQ ID NO: 34) were used.
  • primer 18P-f (5'-GAAGCCGGCAAGCCGGCTTTCACCGCTT-3 ': SEQ ID NO: 35) and primer 18P-r (5'-AAGCAGGGTGAAAGCCCGTCTGCCCGCTCTC-3': SEQ ID NO: 36) were used.
  • primer 38P-f (5'-CTGACCGATGCCCCGTATGACGGGCGTGGT-3 ': SEQ ID NO: 37) and primer 38P-r (5'-ACCACGCCCGTCATACGGGGCATCGGTCAG-3': SEQ ID NO: 38) were used.
  • primer 50P-f (5'-ATGGAGCACAACCCGTACAGATGTCGCGGC-3 ': SEQ ID NO: 39) and primer 50P-r (5'-GCCGGACATCGTACCGGGGTTGTCTCCAT-3': SEQ ID NO: 40) were used.
  • primer 77P-81P-84R-f (5′-CGGTCGCGCCGTCGGTCCACCCCGATCCGCGCGCATCCGCGCGCGCGCGCGGTGCGCGCGCG : SEQ ID NO: 42) was used. PCR was performed using KOD-plus (Toyobo) under the following conditions.
  • the obtained DNA fragment of about 900 bp was subjected to restriction enzyme treatment with NdeI and HindIII, and ligated with pSFN Sm_Aet (International Publication No. 2006/0775486, Examples 1, 6, 12) similarly treated with NdeI and HindIII.
  • pSFN Sm_Aet International Publication No. 2006/0775486, Examples 1, 6, 12
  • this ligation solution E. coli JM109 was transformed, the target plasmid was extracted from the ampicillin resistant strain, and this plasmid was named pSFN-SpAld.
  • One platinum loop of E. coli JM109 / pSFN-SpAld was inoculated into 50 ml of LB liquid medium containing 100 mg / l of ampicillin and shaken at 36 ° C. for 8 hours using a 500 ml Sakaguchi flask.
  • 0.0006 ml of the obtained culture broth was used as a seed liquid medium containing 100 mg / l of ampicillin (glucose 10 g, ammonium sulfate 5 g, potassium dihydrogen phosphate 1.4 g, soybean hydrolyzate 0.45 g as nitrogen content, sulfuric acid
  • ampicillin glucose 10 g, ammonium sulfate 5 g, potassium dihydrogen phosphate 1.4 g, soybean hydrolyzate 0.45 g as nitrogen content, sulfuric acid
  • the seed culture was performed at 33 ° C., aeration 1/1 vvm, stirring 700 rpm, pH was adjusted to 6.3 with ammonia until glucose was consumed.
  • 15 ml of the culture broth thus obtained was added to a main liquid medium containing 100 mg / l of ampicillin (glucose 15 g, ammonium sulfate 5 g, phosphoric acid 3.5 g, soybean hydrolysate nitrogen amount 0.45 g, magnesium sulfate heptahydrate 1 g, iron (II) sulfate heptahydrate 0.05 g, manganese sulfate (II) pentahydrate 0.05 g, thiamine hydrochloride 1 mg, Dis home GD-113K (Nippon Yushi Co., Ltd.) 0.1 ml, pH 6 .3, 0.95 L with water)
  • the mixture was added to a 1000 ml jar fermenter containing 285 ml, and main culture was started.
  • Fractions having aldolase activity were collected and concentrated using Amicon Ultra-15 10k (Millipore). The obtained concentrated solution was diluted with 20 mM Tris-HCl (pH 7.6) to obtain a SpAld solution.
  • the aldolase activity the aldol degradation activity using PHOG as a substrate was measured under the following conditions. Reaction conditions: Phosphate buffer (pH 7.0) 50 mM, PHOG 2 mM, NADH 0.25 mM, MgCl 2 1 mM, lactate dehydrogenase 16 U / ml, 25 ° C., absorbance at 340 nm was measured.
  • OAA DCase Oxaloacetate Decaboxylase from Pseudomonas sp. (Sigma) was used.
  • the enzyme amount (U) used was the value described by the manufacturer.
  • SOD Superoxide Dissimilar from bovine river (Sigma) was used.
  • the enzyme amount (U) used was the value described by the manufacturer.
  • both DAT9 and DAT19 showed a 2R, 4R-monatin accumulation amount higher than that of BMDAT (FIG. 13).
  • Example 6 2R, 4R-monatin one-pot synthesis reaction from L-Trp using DAT9 and DAT19 Using purified DAT, reaction was carried out for 22 hours under the following conditions. The reaction was performed in 1.0 ml using a test tube. DAT was added 1 hour after the start of the reaction. Sampling was performed as appropriate, the sample was diluted with TE buffer, ultrafiltered using Amicon Ultra-0.5 mL centrifugal filter 10 kDa, and the filtrate was analyzed. The analysis was performed by HPLC (same conditions as 4-5-1), and further by HPLC using an optical resolution column for quantification of L-Trp and D-Trp.
  • Reaction conditions L-Trp 20 mM, PA-Na 100 mM, D-Asp 400 mM or D-Ala 400 mM, MgCl 2 1 mM, PLP 50 ⁇ M, Tris-HCl 100 mM, KPB 20 mM, pH 7.6, Ps_aad broth 5%, SpAld 30 U / ml, DAT 1 U / ml, ODC 10 U / ml (when D-Asp is added), SOD 100 U / ml, 25 ° C., 140 rpm.
  • Ps_aad broth was prepared by the following method.
  • the pTB2 strain which is a deaminase expression strain described in International Publication No. 2009/0283338, Example 2, was inoculated into one platinum loop in 50 ml of a TB liquid medium containing 100 mg / l of ampicillin, and 16 ml at 37 ° C. using a 500 ml Sakaguchi flask. Shake for hours. The obtained culture broth was used as Ps_aad broth.
  • Example 7 2R, 4R-monatin one-pot synthesis reaction using modified DAT
  • the enzyme BMDAT (DID-28) modified from BMDAT-22 was evaluated based on structural analysis. Evaluation was performed according to the method described in Example 6, using D-Ala as an amino donor, and 1 U / mL of DAT was added 1 hour after the start of the reaction. Compared with ID22, DID-28 improved the accumulation of 2R, 4R-monatin and D-Trp by-product remained low (FIG. 15).
  • the method of the present invention is useful for producing monatin that can be used as a sweetener.
  • SEQ ID NO: 1 Nucleotide sequence of Bacillus macerans AJ1617-derived dat gene (BMDAT gene)
  • SEQ ID NO: 2 Amino acid sequence of Bacillus macerans AJ1617-derived D-aminotransferase (DAT)
  • DAT D-aminotransferase
  • SEQ ID NO: 3 Bacillus macerans AJ1617-derived D-aminotransferase variant Forward primer for preparation (BmDAT-Nde-f)
  • SEQ ID NO: 4 Reverse primer (BmDAT-Nde-f) for preparing a D-aminotransferase mutant derived from Bacillus macerans AJ1617
  • 5 Forward primer for preparing a D-aminotransferase mutant derived from Bacillus macerans AJ1617 (BmDAT-delNde-f)
  • SEQ ID NO: 6 Reverse primer for preparing a D-aminotransfer

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Abstract

Cette invention concerne : un procédé de production de 2R,4R-monatine à un bon rendement n'utilisant pas de matériau de départ onéreux tel que le D-Trp mais un matériau de départ bon marché, le L-Trp; et autres. Plus spécifiquement, cette invention concerne : un procédé de production de 2R,4R-monatine ou d'un sel de celle-ci, qui comprend les étapes (1) à (3) mentionnées ci-dessous; et autres. (1) Une étape de mise en contact du L-tryptophane avec une désaminase pour obtenir un acide indole-3-pyruvique; (2) Une étape de mise en contact de l'acide indole-3-pyruvique et d'acide pyruvique avec une aldolase pour obtenir 4R-IHOG; et (3) Une étape de mise en contact du 4R-IHOG avec une D-aminotransférase en présence d'un acide D-aminé pour obtenir la 2R,4R-monatine. Dans l'étape (3), il est préférable d'utiliser une D-aminotransférase n'ayant pas la capacité de produire du D-tryptophane à partir de l'acide indole-3-pyruvique ou chez laquelle cette capacité est réduite.
PCT/JP2012/060824 2011-04-25 2012-04-23 Procédé de production de monatine WO2012147674A1 (fr)

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